Unravelling the palaeobiogeographical history of the living fossil genus Rehderodendron (Styracaceae) with fossil and extant pollen and fruit data

Background The relict genus Rehderodendron (Styracaceae), the species of which are restricted to mostly warm temperate to tropical climate in East Asia today, is known from fossil fruits and pollen in Europe during warmer periods from the lower Eocene to Pliocene. To infer which extant species are most closely related to the fossils, new data of pollen and fruit morphologiesy of six extant species, and additional new data of fossil pollen and previously described fossil fruits of Rehderodendron, are compared. Results Both fossil pollen and fruits resemble a morphological mixture of the extant species R. indochinense, R. kwantungense, R. macrocarpum, and R. microcarpum, thus implying that these extant taxa and the fossil European taxa represent an old Eurasian lineage, whereas the pollen and fruit morphology of the extant R. kweichowense and R. truongsonense differ considerably from the fossils and other extant species investigated, and are considered to have evolved independently. Conclusions The palaeobiogeographical history of Rehderodendron reveals that its fossil members of the European lineage were most prominent during climatic optima such as the Palaeocene–Eocene Thermal Maximum (PETM), Early Eocene Climate Optimum (EECO) and Middle Miocene Thermal Maximum (MMTM). However, when during the Pliocene the climate changed to colder and less humid conditions, the genus went extinct in Europe but migrated eastwards, most likely in two dispersal events along the Tethys Sea prior to extinction. One of the former most westerly stepping stones is suggested by the refugial occurrence of R. microcarpum in the southeastern Himalaya, whereas R. macrocarpum and R. kwangtungense, the taxa distributed more to the east, might have migrated eastwards already before the Miocene.


Introduction
The current flora of eastern Asia and southeastern Europe is assumed to be a relict of an ancient Cenozoic flora that thrived along the warm and humid northern margin of the Tethys Sea. The palaeoflora in Eurasia was characterized by a high degree of uniformity [e.g., [1][2][3][4][5] and underwent changes from the Palaeocene to Miocene and Pliocene in response to climatic changes [6,7] and the closing of the Tethys. After the Pliocene, *Correspondence: christa.hofmann@univie.ac.at the thermophilic elements disappeared completely from Europe either prior or during Quaternary glaciation, but many of them still persist in eastern Asia [5]. Manchester et al. [3] considered that some of the extant genera exhibit morphological stasis and therefore can be considered "living fossils" traced back to early Cenozoic times. Rehderodendron appears to be one such fossil, because it was reduced from a widespread geographic distribution during the Cenozoic to only eastern Asia in modern times in response to environmental and climatic changes. Comparable examples are Carya Nuttall (Juglandaceae), which was more diverse in Europe than in North America and Asia during the Neogene, but now has disappeared completely from the modern European flora [8,9], and Styrax Linnaeus (Styracaceae), whose evolutionary history can be traced the through the Cenozoic relictual flora in Europe [10] but today is only present with one species in south east Europe, whereas although it is diverse today in Asia and the Americas.
The Styracaceae family comprises 12 genera and ca. 160-180 woody plant species occurring in warm temperate to tropical climates in the Americas, south Europe, east and south east Asia and Malesia [11,12]. The family forms a well supported clade [10,12], and most of the genera are monotypic or oligotypic with limited geographic distribution, except Styrax, the largest genus [10,13]. Rehderodendron Hu comprises 6-8 species that occur as trees in China, Vietnam and, Myanmar ( Fig. 1; Table 1; [10,11,[14][15][16][17]  Liao; [17]) are considered evergreen and occur in ravine seasonal rain forests or broadleaved montane evergreen forests ( Table 1). The fruit of Rehderodendron is distinguished from those of the other Styracaceae genera by its large size and cylindrical shape, harbouring an endocarp with many irregular rays intruding into the mesocarp [15,17].
The earliest recognizable fossil occurrences of Rehderodendon are fruits of Rehderodendron stonei (Reid & Chandler) Mai from the lower Eocene of England [18,19] and from the middle Eocene of France [20]. Other Styracaceae fossils are fruits of Styrax spp. from the upper Eocene of England [21] and pollen of Styrax from the lower Eocene of Austria [22]. However, because of their characteristic fruit morphology, fossil Rehderodendron fruits have been recognized subsequently from other fossil localities in Europe ranging from Miocene to Pliocene in age. Leaf morphology is of limited utility in distinguishing among Styracaceae genera. No fossil leaves of Rehderodendron have been described.
The overall pollen morphology of Rehderodendron is typical for Styracaceae in general, but until now only a few meagre descriptions and images of extant Rehderodendron pollen exist, mainly in a broader context Table 1 List of extant species investigated (p = pollen; f = fruits), their collection sites, general occurrences and climate according to Köppen-Geiger [44,45] and Köppen-Trewartha [46]  Anther material of Rehderodendron species was soaked in a drop of acetolysis mixture (9:1 acetic acid anhydride: concentrated sulphuric acid) on a glass slide under a binocular and manipulated with a needle to release the pollen from the anthers. The anthers in the acetolysis mixture were repeatedly heated for several seconds over a candle flame to colour the pollen wall and extrude the cell contents. Then the pollen grains were fished out with a micro-manipulator (eyebrow hair mounted on a needle) and transferred to a clean drop of glycerol for LM photography together with a micrometer (Nikon). After photography, the pollen grains were transferred to SEM stubs with a micro-manipulator into minute drops of alcohol to wash off the remaining glycerol, then the stubs were sputter-coated with gold (BIO-RAD) under argon atmosphere and investigated in high vacuum with a FEI Inspect S 500 scanning electron microscope.
The fossil pollen grains were recovered from sediment samples covering the Palaeocene-Eocene-Thermal Maximum (PETM) from England and the Middle-Miocene-Thermal Maximum (MMTM) from Austria and Germany [22,29,31] by treating the sediments with HF and HCL with subsequent acetolysis [e.g., 32,33]. The remaining extracts were mixed with glycerol and smeared onto a glass slides. The manipulation, photography and SEM investigation of fossil pollen followed the same procedures as for the extant pollen. SEM stubs of the fossil pollen are housed in the Department of Palaeontology, University of Vienna under IPUW number 7838a, 7841, 7843. The morphological characters of fossil Rehderodendron fruits were modeled on descriptions in previous studies [18,20,[26][27][28].

Results
Pollen grains of six extant Rehderodendron species were photographed under LM and SEM and their sizes measured ( Table 2, Figs. 2, 3). Depending on the length of heating during the acetolysation process, the pollen changed shape from originally suboblate (unacetolysed state) to subspheroidal and then to more subprolate (fully acetolysed state; Fig. 2). Pollen measurements displayed considerable differences: LM photographs of pollen in glycerol with a micrometer scale yielded larger sizes than the measurements of pollen photographed with SEM (more-or-less desiccated state of pollen after being washed in alcohol, partly desiccated and sputter-coated under argon atmosphere; Table 2, Fig. 2). The measurements of the width and height of the endopori was only possible under LM. However, all measured sizes fall within the size ranges of previously measured Rehderodendron pollen in [23 page 87: R. kwangtungense, R. kweichowense and R. indochinense, the last = is R. macrocarpum according to the junior author] and [24 Table 1: The same is true for the fossil Rehderodendron pollen (see descriptions below).    Remarks: This pollen type comes from the PETM section recovered in exploration drill cores of the London tube in Brixton (England). It was originally affiliated with Ebenaceae (Diospyros in [22]) but clearly is Rehderodendron. It most closely resembles R. kwangtungense and R. macrocarpum.
Remarks: This pollen type has been reported [29 Fig. 3G-I] from the middle Miocene Schaßbach clay pit (Austria; MMTM). It resembles a mixture of R. kwangtungense, R. microcarpum and R. indochinense.

Summarized results of pollen descriptions
The sexine sculpture and ornamentation of R. macrocarpum and R. microcarpum exhibit fluent continuous transitions in the rugulae sizes (larger to smaller); however, R. macrocarpum is more fossulate and less perforate whereas R. microcarpum displays more perforations (Fig. 3). In comparing the sexine of R. microcarpum with R. kwangtungense and R. indochinense the rugulae sizes are also transitional (towards smaller and less pronounced rugulae and more obvious perforations); however R. kwangtungense has more pronounced supratectal micro-gemmae (Fig. 3). Separation among these four taxa is indistinct. Conversely, R. truongsonense, which is conspicuously perforate with supratectal micro-gemmae or echini, and R. kweichowense, which is micro-verrucate to areolate (Fig. 2), can be easily differentiated from each other and the rest of the extant species, and the fossil pollen, which are neither conspicuously perforate and micro-gemmate, nor micro-verrucate to areolate.

Brief descriptions of extant fruits
In general all investigated species develop a thick spongy mesocarp and differ mostly in the rib number and complexity of the endocarp ray system (Fig. 5, Table 3).
The fruits of R. indochinense (Fig. 5A, B) have a characteristic long cylindrical shape with five obvious ribs, and the fruit surface usually has large brown spots, a unique feature among extant Rehderodendron species. The styles are persistent, the stigma is beaked, and the endocarp rays are irregular.
Rehderodendron kwangtungense (Fig. 5C, D) generally is characterized by columnar fruits that are conspicuously ribbed, and the styles are inconspicuous and persistent. The endocarp ray system is complex and displays irregular rays.
The fruits of R. microcarpum (Fig. 5E, F) are more narrower and smaller than all other Rehderodendron fruits with usually an ovoid, cylindrical to fusiform shape and an inconspicuously ribbed surface (5 ribs usually visible). The styles are persistent (conical coracoid). The endocarp has simple, thickened rays.
Rehderodendron macrocarpum (Fig. 5G, H) is characterized by its oblong to elliptic fruits that are conspicuously ribbed (8-12 ribs); persistent styles are very short. The endocarp rays are thick and its rays display irregular thicknesses and lengths.
As mentioned above, the pollen morphology of R. kweichowense and R. truongsonense differ substantially from those of the other four species investigated. This difference is also reflected in their fruits: the fruits of R. kweichowense (Fig. 5I, J) are densely covered with stellate hairs. The fruits also have 10 to12 ribs and irregular endocarp rays. The fruits of R. truongsonense (Fig. 5K, L) are short-terete and inconspicuously ribbed; the endocarp is thickened and comprises an even more complex endocarp ray system than R. macrocarpum.

Comparison of extant and fossil pollen of Rehderodendron
The comparison of LM and SEM images of the three fossil and six extant Rehderodendron pollen described here revealed that the fossil pollen resemble overall four extant Rehderodendron taxa: the lower Eocene Rehderodendron sp. 1. The fossil pollen are always prolate (Fig. 3A-C), which is only the case in extant Rehderodendron pollen when acetolysed for a longer time. The influence of acetolysation under heat is therefore assumed to mimic the fossilization process and the pollen shape can change from suboblate to prolate (Fig. 2A1-F1). 2. As compared to the extant species R. indochinense, R. kwangtungense, R. macrocarpum and R. microcarpum, the rugulae of the fossil Rehderodendron sp. 2 and sp. 3 display a much wider variation in size, and the supratectal arrangement of micro-gemmae on the rugulae in the fossil specimens of Rehderodendron sp. 2 and sp. 3 is generally more diagonally arranged and the individual micro-echini/micro-gemmae are mostly fused into rows (Figs. 2, 3).
However, rugulae size and the degree of fusion of supratectal echini is also various within extant and fossil species. Both, extant and fossil pollen grains show a decrease in rugulae size towards the colpus margo (Fig. 3). There are more than those above: fossil pollen grains were found in Austria and Germany and are of late Oligocene to middle Miocene age [30,[34][35][36][37][38][39] (summary Table 3). They all appear similar to Rehderodendron species sp. 2 and sp. 3 described here and display the same variation of rugulae size and fusion of supratectal micro-gemmae. The  a mix-up of taxa and insufficient images in [23]: their R. macrocarpum is actually R. indochinense and our new images show that R. kweichowense has a completely different tectum ornamentation than the fossil pollen in [30]. Consequently, similar to the other fossil pollen taxa, their pollen taxon resembles a mixture of R. indochinense and R. microcarpum.

Distribution of fossil Rehderodendron fruits and comparison with extant species
Manchester et al. [3,18,[26][27][28]  kwangtungense was the easiest to access and the only one available in European herbaria.
In comparing fruit morphology of fossil Rehderodendron with extant species it can be seen that the fossil fruits do not resemble only R. kwangtungense (Fig. 5C, D) as suggested by Mai [18] and Gregor [24], but other species as well. The smallest fruit is that of R. stonei [3, Fig. 17j-l], displaying as well characters of R. microcarpum (Fig. 5G, H), whereas the medium-sized R. ehrenbergii [e.g., 3, Figs. 45-49; 18, Fig. 17 g-h] more closely resembles R. macrocarpum (Fig. 5E, F), and R. microcarpum (Fig. 5G, H). The largest fruit is that of R. wisaense [18, Fig. 17 [15][16][17] and resembles a poorly developed fruit of R. indochinense (Fig. 5A, B); the same is most likely true for R. dacium and R. custodum (Table 3). We conclude that, like the pollen data, the fossil fruits display the same variation and mixture of morphological characteristics as in extant R. kwangtungense, R. microcarpum, R. macrocarpum, and R. indochinense, and the resemblance to R. indochinense occurs in fossil fruits from the Miocene to Pliocene.

Eurasian distribution of extant and fossil Rehderodendron
The geographic distribution of extant Rehderodendron and its fossil fruits and pollen shows a disjunction up to the Late Miocene (or Pliocene):   Fig. 1) and grow generally in Cwa, Cwb, Cfa climates of the Köppen-Geiger classification ( [44,45]: warm-temperate climate with dry winters and hot or warm summers or fully humid with hot summers) or Cw and Cf climates of the Köppen-Trewartha classification ( [46]: subtropical climate with dry winters or fully humid). The exception is the Vietnamese R. truongsonense [15], which thrives as well under Am climate of the Köppen-Geiger classification ( [44,45]: tropical to subtropical monsoon climate). Fossils similar to R. truongsonense and R. kweichowense are not represented in the fossil record and therefore will be not discussed further. In comparing temperature and rainfall ranges extracted from the climate information based on extant Rehderodendron distribution from WorldClim website (http:// world clim. org/ versi on2; [47], see Table 5), many of the modern species of Rehderodendron appear to easily adapt to subtropical monsoon climate (the annual mean temperature of Rehderodendron ranges from 7.08-19.5 ℃ and annual precipitation range from 893-3856 mm;  [31], Köppen-Geiger data of the MMTM in Austria Lavanttal [30], and CLAMP and Köppen-Geiger data of Schaßbach, Austria [52,53]

The dispersal from Europe to Asia
Fruit dispersal of several taxa by animals and water and the west-east migration during the Eocene from Europe to China and vice versa has been mentioned, amongst other fossil taxa, for Juglans Linnaeus of section Cardiocaryon, Cornus Linnaeus of the blue and white fruited clade, Nyssa sinensis Olivier type, and Symplocos Jacquin subgenus Palura from middle Eocene strata of Hainan [33]. Additionally, a "boreotropical" origin of the entire family of Juglandaceae has been implied by [55], who suggested that Europe played a critical role in the migration and distribution of taxa but also exhibited high extinction of Juglandaceae taxa [55, Fig. 4]. Fitting also . This indicates that water and gravity are important propagation forces for the fruits of this genus and that their thick spongy mesocarp helps them to float in water.
Based on field observations, fruits of Rehderodendron should not float in water for a long time, or else they will rot. Additionally, it has been observed that some rodents (such as squirrels) collect fruits of Rehderodendron for consumption and storage (dyszoochory behaviour), but often destroy the seeds in the fruits and therefore only a small fraction of seeds might be able to germinate in the (forgotten) storage. We therefore suggest that water (and gravity) are the main driving vectors for the lateral fruit distribution and migration of Rehderodendron downslope whereas animals might be responsible for the fruit distribution within the mountain areas.
The dispersal of Rehderodendron to the east may have occurred in three stages.
The existence of a continuous zone of maritime-influenced vegetation along the Tethys during the Cenozoic and the concept of a "boreotropical flora" proposed by Wolfe [5, see also 1, 70] may have played a role in the early dispersal events of Rehderodendron species and many other taxa characteristic of the Eurasian relict flora (see above) (1.) The lower Eocene pollen and fruits from England and the Miocene fossils from Germany and Austria all show similarities, amongst others, with R. kwangtungense. Rehderodendron kwangtungense could be interpreted to represent the oldest developed extant species (Zhao, unpublished data) and might have dispersed eastwards already during middle Eocene times; it  Table 3 Page 15 of 17 Hofmann and Zhao BMC Ecology and Evolution (2022) 22:145 therefore can be found today in the easternmost part of China (see Fig. 1).
(2.) The ongoing Tethys closure and subsequent uplift of the Tibetan Plateau likely hampered the dispersal of Rehderodendron between Europe and East Asia. Additionally, the disappearing Tethys must have shifted the former Eurasian maritime-influenced climate to a more continental monsoonal climate [6, Fig. 2; 7], resulting in the evolution from the Eocene Rehderodendron stonei and Rehderodendron pollen sp. 1 to Rehderodendron ehrenbergii and R. wisaense etc. and to Rehderodendron pollen sp. 2 and sp. 3 during the Miocene (these taxa resemble in part R. indochinense) (3.) The cooling during the Pliocene [6 Fig. 2; 7] caused the extirpation of European Rehderodendron (the extant species of Rehderodendron require annual mean temperatures > 7.08 ℃, and annual precipitation > 893 mm; Table 5). Although the fruits of Rehderodendron are highly variable, it is apparent that fruits of R. ehrenbergii are similar to extant fruits of R. microcarpum, which mostly is distributed in the western part of the distribution of the range of the genus (southern eastern Himalaya; Yunnan and Xizang in China, and Kachin in Myanmar, Fig. 1). If so, then this refugium represents the end of the eastward migration of R. ehrenbergii, however, there is no evidence that the fossil R. ehrenbergii and the extant R. microcarpum belong to the same species. Never-the-less, the southeastern Himalaya was warmer than southeastern Europe during the Pleistocene ice age, which could explain why R. microcarpum survived while R. ehrenbergii perished in Europe. A comparable migration route can be assumed for the Miocene and Pliocene R. wiesaense, R. dacium, and R. custodum the fruits of which resemble mostly the extant R. indochinense, which is distributed slightly farther south of R. microcarpum (Fig. 1).
A somewhat comparable fossil (Eocene and Miocene) and modern distribution pattern to Rehderodendron can be found in Torricellia De Candolle (Torricelliaceae

Conclusions
Fossil pollen grains of Rehderodendron occurred in Europe from the lower Eocene to Miocene and resembles a mixture of characters of the extant R. indochinense, R. kwangtungense, R. macrocarpum, and R. microcarpum. Fossil fruit of Rehderodendron occurred only in Europe from the lower Eocene to the Pliocene (southern Europe) and resemble a mixture of characters of the same species. Fossil pollen grains and fruits with the morphology of R. kweichowense and R. truongsonense are not known and appear to represent a different lineage within Rehderodendron.
Fossil Rehderodendron in Europe grew under warmtemperate to subtropical climate conditions, generally A and C climates of Köppen-Geiger, which is also true for the extant species (China, Vietnam and Myanmar). Fossil Rehderodendron was frequently found in warm and humid periods of the Cenozoic (PETM, EECO, MMTM) and the last warm moist periods of the Pliocene in southern Europe.
Today Rehderodendron is suggested to be an element of the Eurasian "boreotropical" relictual flora which dispersed from Europe to Asia along the Tethys Sea (most probably via water) from the middle Eocene onwards and became extinct in Europe after the Pliocene.